Highly Efficient High-Throughput Transfection
Poster Jul 29, 2008
Markus Zumbansen1, Allison St. Amand2, Devin Leake2, Ludger Altrogge1, and Herbert Müller-Hartmann1
Successful RNAi experiments and large-scale siRNA screens require efficient delivery of highly functional and specific nucleic acids including siRNA oligonucleotides, shRNA vectors, or micro RNAs into an appropriate cell system. Cell types relevant for immunological research, such as primary T cells and several suspension cell lines, are poorly accessible using reagent-based
Nucleofection® is an established method for the effective, non-viral transfection of nucleic acids into difficult-to-transfect cell types including primary cells. With the expansion of the technology to a 96-well format (Fig. 1) highthroughput applications, such as siRNA or shRNA library screenings can now be performed in these cell types rendering target validation and identification possible in cell types highly relevant for medical research. Integration of the 96-well Shuttle® into a liquid handling workstation allows for a fully automated screening approach.
Using the powerful combination of highly functional Dharmacon siGENOME® siRNA reagents and 96-well nucleofection®, we here present data showing the efficient siRNA-mediated gene knockdown in various cell lines and primary cells.
Our studies focus on Jurkat T-cells which are derived from a human acute T-cell leukemia and are extensively used in the study of T-cell signaling and cancer drug development.
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
RNA interference (RNAi) using small interfering RNAs (siRNAs) is an important technology for down-regulation of gene expression and a powerful tool to study cellular processes and pathways. Previously, large collections of siRNAs were available only for traditional experimental model systems, such as human and mouse, and predominantly provided as chemically synthesized libraries.READ MORE